Distance-Hi-C: Creating Photo Activated X-linkers To Define Nuclear Architecture

 描述（由申请人提供）：DNA 的核组织很复杂，由多层组成。在最低分辨率下，染色体的大部分区域堆积在区域中，在较高水平上，染色体区域被组织在拓扑相关的域中，为与启动子和远端调控元件（增强子）结合的转录因子（TF）相互作用提供框架，最后调节子、RNA聚合酶 II 和染色质的结构相互作用导致受调控的基因表达。近年来，人们开发了多种称为染色质（构象）捕获（CC）的方法，并用于捕获构成基因组结构和潜在调控相互作用的动态和稳定的 DNA 接触。这些方法的一个主要限制是它们都依赖于单一交联剂甲醛，该交联剂将 DNA 与蛋白质以及蛋白质与其他蛋白质交联。这使得对观察到的“DNA-DNA”接触的解释变得复杂，并且缺乏距离信息。在这里，我们提出了一种名为 Distance-Hi-C (D-Hi-C) 的正交策略，其中我们设计、测试和应用一组光激活交联剂，旨在直接测量全基因组相互作用位点之间的距离。这些二价交联剂由两个由连接基分隔的反应基团组成。可通过光活化交联的 DNA 嵌入剂（即补骨脂素）将用作反应基团。连接子的精确长度各不相同，本质上是柔性的或刚性的，以便可以推断出交联基因座之间的 3 维距离。这种 DNA 特异性二价交联试剂在 Hi-C 方案中替代甲醛时，会产生空间限制，揭示增强子-启动子相互作用，并可能以前所未有的精度推断核基因组的 3D 排列。此外，D-Hi-C 有望降低背景并允许检查短程和中程相互作用，而这些相互作用被当前方法的高背景所掩盖。除了将生物素掺入到连接产物中之外，在二价交联剂中添加地高辛等基团将能够实现更好的纯化，从而更深入地检查基因组相互作用。最后，基因激活后及时采样 DNA 距离提供了一种探索 4D 架构并在评估基因激活机制时设定关键限制的方法。具体目标是 1) 合成一组二价交联剂并评估它们在体外交联 DNA 的能力； 2) 在果蝇细胞核模型中测试 D-Hi-C 交联剂相对于甲醛的效果； 3) 将新的交联剂应用于 1 级、ENCODE GM12878 和 K562 细胞系，以测试其在人体细胞中的功效； 4) 测试位点特异性光交联，这将允许在基因激活的时间过程中更集中和彻底地检查位点特异性与基因组的相互作用。这些方法的开发将广泛用于提供对基因调控机制的重要见解，这些机制在正常动物发育和体内平衡中起作用，并且在癌症等疾病中出错。

 DESCRIPTION (provided by applicant): Nuclear organization of DNA is complex and consists of multiple layers. At the lowest resolution, large sections of chromosomes are packed in territories, at a higher level chromosomal regions are organized in topologically-associated domains that provide a framework for interactions of transcription factors (TFs) bound to promoters and distal regulatory elements (enhancers), and finally the structural interplay of regulators, RNA polymerase II, and chromatin then lead to regulated gene expression. In recent years, a number of methods called chromatin (conformation) capture (CC) have been developed and used to capture dynamic and stable DNA contacts that constitute genome architecture and potential regulatory interactions. A major limitation of these methods is that they all depend on a single crosslinker, formaldehyde, which crosslinks DNA to proteins as well as proteins to other proteins. This complicates the interpretation of the observed 'DNA-DNA' contacts and it lacks distance information. Here we propose an orthogonal strategy, named Distance-Hi-C (D-Hi-C), where we design, test, and apply a battery of photo-activated crosslinkers, designed to directly measure distances between interacting sites genome-wide. These bivalent crosslinkers consist of two reactive groups separated by a linker. DNA intercalaters that can be crosslinked by photo-activation (i.e. Psoralen) will be used as the reactive groups. Linkers will be of varying precise lengths, and either flexible or rigid in nature so that the 3-dimensional distance between crosslinked loci can be inferred. Such DNA-specific bivalent crosslinking reagents, when substituted for formaldehyde in Hi-C protocol, produces space constraints revealing enhancer- promoter interactions and potentially allowing the inference of the 3D arrangement of the nuclear genome with unprecedented precision. Moreover, D-Hi-C is expected to lower backgrounds and allow examination of short and moderate range interactions, which are obscured by high backgrounds of current methods. Addition of groups such as digoxigenin to the bivalent crosslinkers, in addition to biotin incorporated to the ligation products, will enable better purification and thus deeper examination of genomic interactions. Finally, sampling DNA distances in time following gene activation provides a means of exploring the 4D architecture and setting critical limits in evaluating mechanisms of gene activation. The specific aims are to 1) synthesize a battery of bivalent crosslinkers and evaluate their ability to crosslink DNA in vitro; 2) test D-Hi-C crosslinkers relative to formaldehyde in the Drosophila nuclei model; 3) apply new crosslinkers to tier 1, ENCODE GM12878 and K562 cell lines to test their efficacy in human cells; and 4) test locus-specific photo-crosslinking that will allow a more focused and thorough examination of locus-specific interactions with the genome in a time course of gene activation. The development of these methodologies will be broadly useful in providing critical insights into gene regulatory mechanisms that are operative in normal animal development and homeostasis and that go awry in diseases like cancer.